EFFECT OF PHENYLEPHRINE ON PYRUVATE-DEHYDROGENASE IN FASTING RAT LIVERS

Previous estimates of flux through the pyruvate-dehydrogenase complex were made by measuring (CO2)-C-14 generated from oxidation of [1-C-14]pyruvate, assuming a 1:1 stoichiometry. However, this method fails to discriminate between (CO2)-C-14 produced from pyruvate dehydrogenase and (CO2)-C-14 generated from phosphoenolpyruvate carboxykinase and citric-acid-cycle dehydrogenases. While some previous reports have attempted to correct for the additional (CO2)-C-14 production by comparing (CO2)-C-14 generated by [1-C-14]pyruvate with [2-C-14]pyruvate or [3-C-14]pyruvate, the estimates are flawed by failure to determine the radioactivity and distribution of the C-14 label in the oxalacetate pool. The present method circumvents these problems by utilizing [1,4-C-14]succinate to radiolabel the oxalacetate pool and by directly measuring the specific radioactivity of malate. The results demonstrate that flux through the pyruvate-dehydrogenase complex is negligible compared to the other reactions which generate (CO2)-C-14 from [1-C-14]lactate in the fasted state. Phenylephrine did not significantly alter this result in the fasted state. However, (CO2)-C-14 production via the pyruvate-dehydrogenase complex is large (almost-equal-to 11.5 nmol.min-1.mg mitochondrial protein-1) compared to (CO2)-C-14 production via phosphoenolpyruvate carboxykinase and citric-acid-cycle dehydrogenases (almost-equal-to 6.4 nmol.min-1.mg-1) when the pyruvate-dehydrogenase complex is activated, in the fed state with 1 mM dichloroacetate.